Background We have shown that NRL interacts with TATA-binding proteins (TBP), CRX, NR2E3, SP4 and other transcription factors to control gene expression. Deletion of Nrl in mouse leads to transformation of rods to cones, whereas expression of Nrl in committed cone precursors converts them into rods. A better understanding of the molecular networks that regulate NRL and/or are regulated by NRL and functional characterization of individual key components can provide fundamental insights into photoreceptor biology and dysfunction. &#8232; Results 1. Spatio-temporal gene profiling of rod photoreceptors We are using high throughput photoreceptor gene profiling to construct gene expression networks underlying rod functional maturation and homeostasis. We have taken advantage of a line of transgenic mice, which we generated to express green fluorescent protein (GFP) in newly born and mature rods under control of the Nrl promoter. RNA is extracted from flow-sorted mouse photoreceptor cells and used as template for exon array hybridization. A large amount of data is being analyzed to correlate gene expression changes to differentiation and maturation of photoreceptors, and to their aging and disease. The latter two sets of data will also be analyzed in the context of changes in photoreceptor function in aging and diseased photoreceptors (see project # EY00475-02). 2. Genes/pathways that guide rod differentiation and homeostasis (NRL targets) 2.1. We have employed in silico and chromatin immunoprecipitation (ChIP) followed by very high throughput sequencing (ChIP-seq) methods, in combination with Nrl-knockout gene profiles, to identify direct transcriptional targets of NRL in mature mouse rods. A number of candidates were confirmed by ChIP-qPCR and tested functionally by in vivo RNAi-mediated knockdown experiments in the developing mouse retina. Enhancer analysis was performed to identify candidate NRL co-activators/co-repressor partners on selected targets. 2.2. Among NRL targets is the rod-specific gene rhodopsin. Rhodopsin is a major phototransduction protein, and misregulation of its expression results in retinal diseases such as retinitis pigmentosa. We are elucidating transcription regulatory proteins that positively or negatively control rhodopsin expression. An analysis by mass spectrometry of individual components bound to different regions of the rhodopsin promoter has revealed the presence of several new proteins. Not surprisingly, a number of proteins are involved in splicing or chromatin remodeling. After validation by immunoblot analysis, we are characterizing three proteins that synergistically enhance NRL and CRX activity on the rhodopsin promoter. To delineate the mechanism of delay in rhodopsin expression during rod maturation, we are examining possible negative regulators of the rhodopsin promoter. We have identified evolutionarily conserved regions and performed in vivo electroporation and reporter gene assays to show that the most proximal conserved promoter region is sufficient to sustain reporter gene activity even at postnatal day (P)2. However, the region -174 to -1610 likely contains elements that negatively regulate rhodopsin expression in immature photoreceptor cells. In silico analysis combined with gene expression data identified putative transcription factors that bind in that region. Selected candidate negative transcription regulators of rhodopsin gene expression are being tested further. 3. Pathways upstream of NRL (photoreceptor cell fate specification) 3.1. To understand the regulation of Nrl expression during photoreceptor differentiation and in the mature retina, we are investigating cis-elements in the Nrl promoter and their cognate regulatory binding factors. We found that, upon in vivo electroporation, conserved sequences in the first and second proximal conserved regions (comprising a 900 bp promoter sequence) are responsible for sustained GFP expression specifically in rods. Furthermore, a minimal promoter sequence comprising the TATA like binding element and a second proximal conserved region are sufficient to drive specific GFP expression in rods. By electrophoretic mobility shift assay (EMSA), we have identified transcription factors that differentially bind to NRL conserved promoter regions in developing and mature mouse retina. Transcriptional activity of selected proteins on NRL minimal promoter is being tested by in vitro luciferase assays. 3.2. We have established that the transcriptional activity of NRL is modulated by di-sumoylation at the Lys-20 residue, a site that is conserved in NRL across species and in proteins of the MAF family (that includes NRL) (Ref. 3). NRL-K20R and NRL-K20R/K24R sumoylation mutants show reduced transcriptional activation of Nr2e3 and rhodopsin promoters (two direct targets of NRL) in reporter assays when compared with wild-type NRL. Consistent with this, in vivo electroporation of the NRL-K20R/K24R mutant into newborn Nrl-knockout mouse retina leads to reduced Nr2e3 activation and only partial rescue of the Nrl-knockout phenotype, in contrast to wild-type NRL that can convert cones to rods. Although PIAS3 (protein inhibitor of activated STAT3), an E3-SUMO ligase implicated in photoreceptor differentiation, can be immunoprecipitated with NRL, there appears to be redundancy in E3 ligases, and PIAS3 does not seem to be essential for NRL sumoylation. This differentiates NRL from NR2E3, which is modulated by PIAS3-mediated sumoylation. These data suggest that gene regulatory networks regulating cell fate in the retina not only include transcription factors and their target genes, but also an array of proteins that can modulate the activity of the transcription factors by post-translational modification, most likely in response to extracellular signalling pathways. To further investigate the role of PIAS3 in retinal development, PIAS3 floxed mice have been generated and are being bred with Rx-Cre (for deletion in all retinal cells) and with Crx-Cre (for photoreceptor specific deletion) mice to establish the role of SUMOylation in regulating NRL function. Preliminary data have also indicated that NRL stability is regulated by GSK3-mediated phosphorylation. GSK3 is a regulator of retinal progenitor differentiation and synaptogenesis. Deletion of GSK3a and/or GSK3b specifically in all retinal progenitor cells leads to a large increase of displaced ganglion cells (in the INL) and abnormal synaptogenesis. As compound GSK3a and GSK3b cKO with Rx-Cre is lethal, we are currently generating double KO mice using Six3-Cre and Crx-Cre. 4. Effects of NRL mutations on photoreceptor development and homeostasis The transgenic mice that we generated to express human NRL S50T and NRL P51S mutations in the Nrl-KO background underwent normal retinal development and rod differentiation. No abnormal histological or functional (ERG) phenotype could be observed up to 12 months of age. However, upon short exposure (1 hr) to bright light, almost complete rod, but not cone, degeneration was observed even at young ages. The phenotype appeared to be modified by the genetic background. S50T and P51S mutations prevent NRL phosphorylation and increase its transcriptional activity on the rhodopsin promoter. Although it appears that NRL phosphorylation is dispensable for rod differentiation it is essential for rod maintenance upon tissue damage. &#8232; Significance &#8232;As our studies identify novel genes and networks that control photoreceptor development and function, we are gaining new insights into photoreceptor biology and disease. Components of the regulatory networks can be modulated for treatment of retinal diseases. Additionally, our research will greatly assist in designing possible use of stem cells for therapies of degenerative diseases that involve photorecetpor dysfunction/death.